Human immunodeficiency virus type 1 (HIV-1) is a member of the lentivirus subfamily of retroviruses. Like all retroviruses, lentiviruses encode the gag, pol, and env genes. However, lentiviruses also contain several accessory genes. The accessory gene vpu, which is unique to HIV-1, encodes viral protein U (Vpu) (44). Vpu is a 16-kDa type I integral membrane phosphoprotein that can form oligomeric structures in vitro and in vivo (32, 43). Indirect immunofluorescence indicates that Vpu localizes predominantly to the Golgi complex (29), but some Vpu is also present in association with the plasma membrane (17). The protein contains a hydrophobic N-terminal domain, which serves as the membrane anchor, and a C-terminal hydrophilic cytoplasmic domain (32).
Vpu plays two roles in HIV-1 replication. First, Vpu promotes the specific degradation of the HIV-1 receptor, CD4, in cell-free systems (8) and in vivo (41, 47). Degradation of CD4 enhances the transport and subsequent processing of the HIV-1 envelope glycoprotein by releasing it from complexes with CD4 that trap both proteins in the ER (28). Direct interaction of Vpu with the cytoplasmic domain of CD4 is required, but not sufficient, for CD4 degradation (4). Mutational analysis indicates that the hydrophilic cytoplasmic domain of Vpu is required for Vpu-mediated CD4 degradation (40). The second function of Vpu is the enhancement of virus particle release (19, 29, 43, 45, 48). The effect of Vpu on virus particle release appears to be mediated from a post-ER compartment (41). Whereas the cytoplasmic domain of Vpu is important for the degradation of CD4, the transmembrane domain of Vpu is sufficient for partial enhancement of virus release (40). Thus, based on both differential intracellular site of action and genetic criteria the bipartite roles of Vpu are mechanistically distinct. The HIV-1 Gag protein is sufficient for immature virus capsid formation, and those capsids are fully competent for Vpu-mediated enhancement of release, indicating that an eventual target of Vpu during particle release is intrinsic to Gag (30).
The identification of host cell proteins that function in HIV replication has provided crucial insight into the intricacies of the biology of HIV-1. The identification of CD4 as the principal virus receptor on T-cells has provided a basic paradigm for virus entry (10). Chemokine receptors are proteins involved in chemotaxis of immune system cells and have been co-opted by HIV-1 to allow entry into host cells in conjunction with CD4 (2, 13). Urokinase-type plasminogen activator, a proteinase involved in tissue invasion by macrophages, binds to and cleaves the HIV-1 envelope glycoprotein gp120 and enhances the infectivity of HIV-1 in macrophages (24). Cyclophilins are proteins that bind to the immunosuppressive drug cyclosporin A (CsA) and are members of the immunophilin superfamily, which includes members that facilitate protein folding (18). Cyclophilins A, B, and C interact with HIV-1 Gag, and cyclophilin A is incorporated into virions (15, 31, 46). The incorporation of cyclophilin A into virus particles is required for an early step in replication between membrane fusion and reverse transcription (5). Furin, a subtilisin-like endoprotease, mediates the cleavage of the HIV-1 envelope glycoprotein precursor gp160 to gp120 and gp41, a process required for virus infectivity (22).
In a preferred embodiment, the present invention is an assay suitable to determine inhibitors or modulators of the Ubp/Vpu interaction. This assay comprises the steps of exposing Vpu and Ubp together in the presence of a candidate inhibitor under conditions in which Vpu and Ubp would normally interact in the absence of the inhibitor. One then determines whether Vpu and Ubp interaction occurs. This assay may be either in vivo or in vitro. A successful inhibitor or compound that alters Vpu-Ubp interaction is an excellent candidate for an HIV therapeutic. Conversely, in some instances an increase in affinity might negatively affect replication.
The present invention is also an assay for determining whether a candidate inhibitor inhibits the interaction between Gag and Ubp. This assay comprises the steps of exposing Ubp and Gag in the presence of a candidate inhibitor under conditions in which Ubp and Gag would interact in the absence of the inhibitor. One then determines whether Gag and Ubp interact. This assay may be either in vivo or in vitro. As with the assay above, successful inhibitors or compounds that affect interaction would make excellent candidates for anti-HIV therapeutics.
In another embodiment the present invention is a preparation of U binding protein (Ubp).
In another embodiment, the preparation is a gene sequence encoding Ubp.
In another embodiment, the preparation is an anti-Ubp antibody. This antibody may be either monoclonal or polyclonal.
It is a feature of the present invention that an assay is provided that provides candidates for HIV therapeutics.
It is another feature of the present invention that a preparation of U-binding protein and a gene sequence encoding U-binding protein is disclosed.
Other features, objects and advantages of the present invention will become apparent after examination of the specification, claims and drawings.